The composition of the plant cell wall is complex and variable. Polysaccharides are mainly found in the form of long chains of cellulose (the main structural component of the plant cell wall), hemicellulose (comprising various .beta.-xylan chains) and pectic substances (consisting of galacturonans and rhamnogalacturonans; arabinans; and galactans and arabinogalactans). From the standpoint of the food industry, the pectic substances, arabinans in particular, have become one of the most important constituents of plant cell walls (Whitaker, J. R. (1984) Enzyme Microb. Technol., 6, 341).
Arabinans consist of a main chain of .alpha.-L-arabinose subunits linked .alpha.-(1.fwdarw.5) to one another. Side chains are linked .alpha.-(1.fwdarw.3) or sometimes .alpha.-(1.fwdarw.2) to the main .alpha.-(1.fwdarw.5)-L-arabinan backbone. In apple, for example, one third of the total arabinose is present in the side chains. The molecular weight of arabinan is normally about 15 kDa.
Enzymes capable of degrading arabinans are becoming increasingly important to the food industry. In juice production, for example, the demand to increase yields in order to reduce production costs has necessitated the modification of traditional processes. The utilization of enzymatic pre-treatments of the fruit pulp before pressing with specific enzymatic products drastically improves the juice yield by solubilizing the cell wall polysaccharides.
However, a persistent turbidity commonly referred to as "arabinan haze" has been a source of problems in the production of concentrated juices. The arabinan haze is more often present in concentrated juice than in non-concentrated juice. This may indicate that water activity has an influence on the solubility of arabinan. Furthermore, it has been found that this haze is soluble between 60.degree. and 80.degree. C.
It has also been found that while branched arabinan is soluble in concentrated chilled apple and pear juices, the linear, debranched .alpha.-(1.fwdarw.5)-L-arabinan is much less soluble. This debranched .alpha.-(1.fwdarw.5)-L-arabinan is formed from the L-arabinan by the action of an arabinan-degrading enzyme present in the commercial pectic enzyme preparations from Aspergillus niger, commonly used to increase juice yield after pulp treatment but before pressing.
On the other hand, debranched arabinans are considered desirable for certain other applications. WO 90/06343 discloses the debranching of sugar beet araban by the action of an .alpha.-L-arabinofuranosidase, free of endo arabinanase activity, which is isolated from a culture filtrate of Aspergillus niger or from a commercial pectinase mixture using ion-exchange and gel filtration chromatography procedures. The debranched araban may be used as a fat substitute in foods.
Arabinan-degrading enzymes are known to be produced by a variety of plants and microorganisms, among these, fungi such as those of the genera Aspergillus, Corticium, Rhodotorula (Kaji, A. (1984) Adv. Carbohydr. Chem. Biochem., 42, 383), Dichotomitus (Brillouet et al. (1985) Carbohydrate Research, 144, 113), Ascomycetes and Basidomycetes (Sydow, G. (1977) DDR Patent Application No. 124,812).
In particular, the filamentous fungus Aspergillus niger is known to produce three different arabinan-degrading enzymes: an .alpha.-L-arabinanase having a molecular weight of approximately 35 kDa and two .alpha.-L-arabinofuranosidases having molecular weights of approximately 118 and 60 kDa, respectively, (Rombouts et al. (1988) Carbohydrate Polymers, 9, 25). N. B. van der Veen et al. ((1991) Arch. Microbial., 157, 23) reports molecular weights of 43, 83 and 67 kDa for these same three enzymes, respectively.!
The 35 kDa arabinanase (also known as ABN A) has endo activity and exclusively cleaves 1.fwdarw.5 linkages. The activity of this enzyme decreases as the 1,5-.alpha.-L-arabinan sequences become shorter and the concentration of arabinose dimers and trimers increased (Rombouts et al., supra). N. B. van der Veen et al. (1991) reported a molecular weight of 43 kDa.!
The 118 kDa .alpha.-L-arabinofuranosidase (also known as arabinofuranosidase A, ABF A or EXO A) exclusively cleaves 1.fwdarw.5 linkages with exo-type activity as shown by the accumulation of arabinose monomers. This enzyme displays the highest activity on low molecular weight substrates (Rombouts et al., supra). N. B. van der Veen et al. (1991) reported a molecular weight of 83 kDa.!
The 60 kDa .alpha.-L-arabinofuranosidase (also known as arabinofuranosidase B, ABF B or EXO B), also having exo-type activity, predominantly cleaves 1.fwdarw.3 and 1.fwdarw.2 .alpha.-L-arabinans, yet also demonstrates the ability to cleave 1.fwdarw.5 .alpha.-L-arabinans. Again, only arabinose monomers are detected after degradation of an .alpha.-L-arabinan-containing substrate (Rombouts et al., supra). N. B. van der Veen et al. (1991) reported a molecular weight of 67 kDa.!
The A. niger ABF B enzyme has also demonstrated the ability to cleave the 1.fwdarw.6 linkage between a terminal arabinofuranosyl unit and the intermediate glucose of monoterpenyl .alpha.-L-arabinofuranosylglucosides (Gunata et al. (1989) European Patent Application No. 332.281; Gunata et al. (1990) J. Agric. Food Chem., 38, 772).
Enzymes having an activity similar to that of the A. niger ABF B enzyme have been identified in other fungi such as Dichotomitus squalens (Brillouet et al., supra), Corticium rolfsii and Rhodotorula flava (Kaji, A., supra).
A possible solution to the problem of haze formation in juices may be to use an enzyme formulation having an improved balance between the endo arabinanase (ABN A) and the ABF A and ABF B enzymes, or alternatively, enzymes having similar activities, i.e. one or more arabinofuranosidases obtained from another microorganism which is capable of cleaving 1.fwdarw.3 and 1.fwdarw.2 .alpha.-L-arabinans, as well as 1.fwdarw.5 .alpha.-L-arabinans. However, normal fermentation of A. niger fails to yield sufficient levels of the desired arabinofuranosidases. Moreover, it would be advantageous to be able to attenuate the amount of ABF A, ABF B and ABN A activities for optimal results in specific applications.
The ability of the ABF B enzyme to cleave the terminal 1.fwdarw.6 linkage of monoterpenyl .alpha.-L-arabinofuranosylglucosides may be used to assist in the release of aroma components from various fruit juices and thus, the enhancement of their flavors (Gunata, Z. et al. (1989 and 1990), supra). However, it would be preferable to use a purified ABF B or ABF B-like enzyme for this purpose since the presence of other enzymatic activity may degrade other important components of the juice and, in so doing, have a detrimental effect on the ultimate quality of the juice.
In nature, microbial arabinan-degrading enzymes are always produced together with other enzymes having polysaccharide-degrading activities, such as pectinases, xylanases, acetyl xylan esterases, acetyl esterases, galactanases and cellulases. As mentioned above, for some applications, these enzyme activities are not needed or are unwanted. Moreover, due to relatively low expression levels in the wild-type strains, the arabinan-degrading enzymes have proven to be somewhat difficult to isolate both from each other and from other enzymes produced by A. niger.
It is known that fermentation conditions may be varied to favor the production of an enzyme of interest. It is also known that the cloning of the gene encoding the desired enzyme and overexpressing it in its natural host, or in another compatible expression host will specifically enhance the production of the enzyme of interest. This latter method is particularly useful if the enzyme of interest is to be obtained in increased amounts, or in a form which is free of other, undesired enzymatic activity.
Clearly, it would be useful to increase arabinan-degrading activity via recombinant DNA techniques. However, until now, the genes encoding the A. niger arabinan-degrading enzymes have not been available. Accordingly, it would be of great importance to obtain genes encoding arabinan-degrading enzymes of fungal origin which may be brought to expression in its native host or, alternatively, in other microbial hosts wherein high expression levels of one or more arabinan-degrading enzymes may be achieved.
The expression of recombinant bacterial .alpha.-L-arabinofuranosidase has been previously described by Schwarz et al. ((1990) Biochem. Biophys. Res. Commun., 170, 368). The gene encoding a bacterial arabinofuranosidase was isolated from Clostridium stercorarium and brought to expression in an E. coli host.
A gene encoding an arabinosidase was cloned from the anaerobic bacterium Bacteriodes ovatus and brought to expression in E. coli, as disclosed by Whitehead & Hespell ((1990) J. Bacteriol., 172, 2408).
However, E. coli expression hosts are, in some instances, considered to be unsafe for the production of proteins by recombinant DNA methods due to their production of unacceptable by-products such as toxins. Furthermore, in E. coli expression, large amounts of heterologous protein tend to accumulate inside the cell. Subsequent purification of the desired protein from the bulk of E. coli intracellular proteins can sometimes be difficult.
Moreover, since bacterial genes contain no introns, one is confronted with few problems in cloning and expressing such genes in prokaryotic hosts. On the other hand, the expression of eukaryotic genes is not always so straightforward. It is well known that genes isolated from eukaryotic strains may contain introns. This inherently introduces complications in the cloning and expression of these genes, should a prokaryotic host be preferred.
Furthermore, certain differences exist between the physical characteristics of arabinan-degrading enzymes of fungal origin and those from bacteria. In general, fungal enzymes have a pH optimum in the range from .ltoreq.3.0-6.0. A few fungal species produce arabinan-degrading enzymes having pH optima as low as pH 2.0. These pH optima are generally significantly lower as compared to similar enzymes from bacterial strains which have a pH optimum in the range of pH 5.0-7.0 (Karimi and Ward (1989) J. Indust. Microbiol., 4, 173; Lee and Forsberg (1987) Can. J. Microbiol., 33, 1011). Thus, it is clear that bacterial arabinan-degrading enzymes are less suitable for use in, for example, processes requiring lower pH conditions.